Efficacy of Lodoxamide Eye Drops on Mast Cells and Eosinophils after Allergen Challenge in Allergic Conjunctivitis

Stefano Bonini, MD,U Mauro Schiavone, MD, 1 Sergio Bonini, MD,3 Laura Magrini, MD,4

Pietro Lischetti, MD/ Alessandro Lambiase, MD/ Massimo G. Bucci, MD1 •2

Purpose: The purpose of the study is to evaluate in a double-blind, randomized study the efficacy of lodoxamide tromethamine 0.1% versus placebo.

Methods: Signs and symptoms, tear tryptase, and tear fluid cytology were evaluated in 20 asymptomatic subjects with allergic conjunctivitis. The study included three allergen challenges in skin test-positive patients. At the first visit, a threshold dose of allergen was established. At the second visit, a bilateral ocular challenge was performed without pretreatment. At the third visit, either lodoxamide or placebo eye drops were used for 1 week before ocular challenge.

Results: Lodoxamide significantly reduced tryptase levels (P < 0.01 ), neutrophils (P < 0.04), and eosinophils (P < 0.01) in the tear fluid and significantly inhibited ocular itching (P < 0.02) when compared with that of placebo.

Conclusions: Lodoxamide is effective in reducing tryptase levels and the recruit­ment of inflammatory cells in the tear fluid after allergen challenge. Ophthalmology 1997; 104:849-853

Allergic conjunctivitis is a common seasonal or perennial ocular disease characterized by the presence of itching, burning, photophobia, and redness of the conjunctiva. Al­though the cornea generally is spared from the infiarnma-

Originally received: May 15, 1996. Revision accepted: January 8, 1997. 1 Department of Ophthalmology, University of Rome "Tor Vergata," Rome, Italy. 2 G. B. Bietti Eye Foundation, Rome, Italy. 3 Clinical Immunology and Allergology, University of Naples, Naples, Italy. 4 Institute of I Clinica Medica, University of Rome "La Sapienza," Rome, Italy.

Presented at the ARVO Annual Meeting, Ft. Lauderdale, FL, April 1996.

The anthers have no proprietary interest in the development or marketing of this or a competing drug.

Address reprint requests to Stefano Bonini, MD, Via Cassia Antica, 35, 00191 Rome, Italy.

tory process and vision is not affected, the symptomatic phase of the disease is responsible for patient discomfort and requires adequate treatment. Allergic conjunctivitis is a type I immediate hypersensitivity disease in which the contact of immunoglobulin-E-bound on the mast cells with the offending allergen is responsible for the ocular signs and symptoms. 1 Consequently, mast cell stabilizers and antihistamines have been used successfully for many years to reduce the ocular signs and symptoms of this condition. 2

Recent advances in understanding the pathogenesis of allergic conjunctivitis have been obtained by means of the conjunctival provocation test (CPT) with the sensitizing allergen.3 Conjunctival provocation test is a reproducible and rapid test that mimics allergic conjunctivitis in hu­mans. It has been used clinically to study the irnmunopa­thogenesis of allergic conjunctivitis by evaluating the tear fluid cell profiles and by measuring chemical mediators of allergic infiammation.4

•5 This test can be used as an

indicator of the efficacy of topical antiallergic drugs.6

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Table 1. Characteristics of Subjects with Allergic Conjunctivitis

Duration of the

Age Disease Threshold Dose (BU/ml) Allergen Used Patient No. (yrs) Sex Skin Test and/or RAST (yrs) Family History (both eyes) for CPT

Lodoxamide tromethamine 0.1 o/o 1 43 M Grass 3 Negative OD 32,000, OS 1,000 Grass 2 16 M Grass, Dpt 7 Positive ou 10,000 Grass 3 26 F Par, Grass 1 Positive ou 100,000 Grass 4 24 M Grass 3 Negative ou 10,000 Grass 5 33 F Grass 10 Positive ou 320,000 Grass 6 29 M Par 1 Positive ou 3200 Par 7 17 F Grass, Par, Dpt 6 Negative ou 100,000 Grass 8 17 F Grass, Par, Dpt 10 Positive ou 1000 Grass 9 30 M Grass 20 Positive ou 100,000 Grass

10 25 M Grass, Par, Dpt 12 Negative ou 1000 Par 11 34 F Par, Grass 16 Negative ou 1000 Par 12 35 M Par, Grass 3 Positive ou 10,000 Par Placebo 13 24 M Grass 12 Negative ou 320,000 Grass 14 39 F Par 3 Positive ou 3200 Par 15 22 F Par 3 Positive ou 32,000 Par 16 26 M Grass, Dpt 17 Positive ou 100,000 Grass 17 15 M Par 7 Negative ou 3200 Par 18 18 F Grass, Par, Dpt 15 Positive ou 100,000 Par 19 8 M Par 8 Negative ou 32,000 Par 20 23 F Par, Dpt, Grass 14 Positive ou 32,000 Par

M = male; F = female; RAST = radioallergosorbent test; Grass = Rye grass; Par = Parietaria officinalis; Dpt = Dennatophagoides pteronyssimus; OD = right eye; OS = left eye; OU = both eyes; CPT = conjunctival provocation test.

Lodoxamide tromethamine 0.1% is a newly developed antiallergic compound administered in the form of eye drops. Lodoxamide tromethamine has been shown to re­duce the ocular signs and symptoms in subjects with ver­nal conjunctivitis and allergic conjunctivitis, presumably by inhibiting mast cell degranulation and the toxic effects of eosinophil-derived products on corneal epithelium?- 10

However, no objective measurements of the effect of this compound on the inflammatory cell profile or mast cell­derived products in tears have been made in human aller­gic conjunctivitis.

In the current double-blind, randomized study, we evaluated the efficacy of lodoxamide tromethamine 0.1% eye drops in 20 asymptomatic subjects with allergic con­junctivitis induced by the CPT with the sentizing allergen. We also measured the level of a mast cell-derived media­tor, tryptase, and counted the inflammatory cells in the tear fluid of subjects with allergic conjunctivitis before and after ocular challenge.

Patients and Methods

Patients

Twenty asymptomatic subjects with allergic conjunctivi­tis were included in this double-blind, randomized study. Informed consent was obtained from all subjects. There were 11 men (mean age, 25.0 ::±:: 9.6 years) and 9 women (mean age, 25.4 ::±:: 8.1 years) (Table 1). The diagnosis of

850

allergic conjunctivitis was based on seasonal or chronic history of symptoms related to allergic condition and on positive skin test results or radioallergosorbent test to common allergens (Dermatophagoides pteronyssinus, Parietaria officinalis, and Rye grass). Inclusion criteria for subjects to enter the study included:

1. Males or females aged 8 to 50 years with an history of at least 1 year of allergic conjunctivitis.

2. Sensitization detected by prick test or radioallergo­sorbent test to at least one allergen.

3. Absence of signs and symptoms of allergic conjunc­tivitis at the time of the study.

4. Patients without any ocular or systemic disease. 5. Patients without any topical or systemic treatment

from at least I week before entering the study.

Patients with other ocular or systemic diseases or treated with systemic steroids, antihistaminic, or specific immunotherapy; lactating or pregnant women; or subjects wearing contact lenses were excluded from the study.

Study Design

Three consecutive CPTs were performed for each patient 1 week apart. The allergen used for CPT were those that gave the greatest positive response to skin test or radioal­lergosorbent test (Table 1). The first CPT was used to establish the dose of allergen that induced a clinical score

Bonini et al · Lodoxamide in Allergic Conjunctivitis

of conjunctival hyperemia of 2+. The conjunctival hyper­emia was evaluated and scored as follows: 0, no reaction; 1 +,mild; 2+, moderate; 3+, severe with mild chemosis; 4+, very severe with intense chemosis of the conjunctiva and edema of the lid. Briefly, one drop of standardized allergen was dropped into the lower conjunctival fornix of both the eyes starting from the lowest dilution avail­able. There were six dilutions of allergen (1000; 3200; 10,000; 32,000; 100,000; 320,000 BU/ml of albumin; Pharmalgen, Pharmacia Diagnostics AB, Uppsala, Swe­den). If no conjunctival reaction occurred in 5 minutes, the next higher dose was tested until a minimum of 2+ score of hyperemia was reached. This dose was consid­ered the individual threshold dose and was used in the second and third CPTs. The second CPT was performed in both eyes 1 week later. This test was used to assess the reproducibility of the conjunctival hyperemia. The third CPT was performed after a week of topical treatment with lodoxamide tromethamine 0.1% ophthalmic solution or placebo in both eyes.

The following parameters were considered to evaluate the conjunctival response before and 5 minutes after the second and third CPTs: (1) clinical data, (2) tear fluid cytologic analysis, (3) tear tryptase determination, and (4) patient and physician opinions.

Clinical Data. Hyperemia, itching, burning, tearing, grittiness, and chemosis of the conjunctiva were evalu­ated. A photograph of conjunctival hyperemia also was taken at each time period. Each conjunctival sign and symptom and the photographs were evaluated by two masked investigators and scored as follows: 0, no reac­tion; 1 +, mild reaction; 2 +, moderate reaction; 3 +, se­vere reaction; and 4+, very severe reaction.

Tear Fluid Cytologic Analysis. Two microliters of tears were collected with a microcapillary tube (Sigma, St. Louis) from each eye at each time of observation. Tears were spread on a glass slide, fixed, and stained (Diff-Quick; Baxter Healthcare, Gibbstown, NJ) as de­scribed previously. 10

•11 The cells present in each slide

were counted and examined at a magnification of X 100 by light microscopy.

Tear Tryptase Determination. Ten microliters of tear fluid were collected with a microcapillary tube (Sigma) from the inferior conjunctival fornix at each time period and stored at -20° C until assayed. Tear tryptase levels were measured by a sandwich solid-phase radioimmuno­assay (Tryptase RIACT; Kabi Pharmacia, Uppsala, Swe­den) based on the reaction between a monoclonal anti­body linked to the solid phase of a test tube with the tryptase present in samples. The solid phase is then re­acted with a second monoclonal antibody tagged with the radioactive isotope of iodine (1125

). Tear fluid was diluted 1:5. The lower limit of sensitivity of the test with 50 J.Ll of sample was shown to be 0.5 J.Lg!l.

Patient and Physician Opinions. Overall patient and physician opinions, in terms of pretreatment efficacy, ob­tained after the second and third CPTs were recorded and graded as follows: 1 +, greater conjunctival signs and symptoms in the second CPT; 2+, no difference in con­junctival signs and symptoms after the second and third

CPTs; 3+, greater conjunctival signs and symptoms in the third CPT.

Results were evaluated and compared in the second and third CPTs for statistical analysis. A nonparametric test (Wilcoxon signed-rank) was used to compare sub­jects' treated eyes with placebo-treated eyes. A probabil­ity of 0.5 or less was accepted as statistically significant.

Results

All the allergic subjects showed an ocular hypersensitivity reaction after allergen challenge. However, different threshold doses were needed to induce a 2+ conjunctival hyperemia. Rye-grass (10 subjects) and Parietaria offici­naZis allergens were used for conjunctival provocation (Table 1). The reproducibility of the test was confirmed in the second CPT. Twelve subjects received lodoxamide tromethamine 0.1% eye drops (7 men, 5 women; mean age, 27.4 ::'::: 8.2 years) and 8 received placebo (4 men, 4 women; mean age, 21 ::'::: 9 years) (Table 1).

Lodoxamide tromethamine 0.1% ophthalmic solution significantly reduced (P < 0.02) the ocular itching in­duced by allergen challenge when compared with that of placebo (median score ::'::: standard error was, respectively, 0 ::'::: 0.1 and 1 ::'::: 0.1 in the lodoxamide and in the placebo pretreated eyes). According to our grading system, no significant differences were observed in the clinical score comparisons of conjunctival hyperemia, burning, tearing, grittiness, chemosis, and in the photographic evaluation between the lodoxamide-treated group and the placebo­treated group.

Neutrophils (P < 0.04) and eosinophils (P < 0.01) were reduced significantly in the tear fluid after treatment with lodoxamide when compared with that of placebo (Fig 1). Similarly, tear tryptase levels (P < 0.01) were lower in the lodoxamide-treated group when compared with those of placebo (Fig 2). Patients', but not physi­cians', overall opinions were significantly (P < 0.02) in favor of the lodoxamide treatment (median score ::'::: stan­dard error of subjects' opinions were, respectively, 1.5 ::'::: 0.1 and 2 ::'::: 0.1 in the lodoxamide and in the placebo pretreated eyes). No ocular or systemic side effects were induced by ocular challenge with allergen.

Discussion

Lodoxamide tromethamine is a newly developed antial­lergic agent that was found to inhibit passive anaphy­laxis and bronchoconstriction in patients with asthma after specific challenge. 11 After ocular application, lo­doxamide inhibited the allergic conjunctival reaction and reduced the release of histamine in an experimental model of allergic conjunctivitis in the rat. 8 Lodoxamide also reduced the clinical signs and symptoms of both vernal keratoconjunctivitis and allergic conjunctivitis in humans.7

-10 We found that lodoxamide significantly

reduced the ocular itching induced by allergen chal­lenge. Although it is supposed that lodoxamide exerts

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Ophthalmology Volume 104, Number 5, May 1997

a protective effect on mast cells and prevents the toxic effects induced by eosinophils on the corneal epithe­lium, there is no evidence of objective decrease of in­flammatory cells or mast cell mediators in human tears of subjects with allergic conjunctivitis.

Tryptase is a neutral endoprotease present in the secre­tory granules of human mast cells that has been used as a specific assessment of their activation. Tryptase is a diagnostic marker unique to mast cells, whereas histamine also is produced by other cells. 12

'13 Accordingly, different

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• • • • •

• • • • I •

Lodoxamide

•

••••••••••• Lodoxamide

• •

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•

• •

• •

• • • Placebo

Figure 1. Neutrophils and eosinophils in 2 J.Ll of tear fluid 5 minutes after allergen challenge. The lodoxamide-treated eyes showed a significant reduction in the number of neutrophils and eosinophils (respectively, P < 0.04 and P < 0.01) when compared with that of placebo.

852

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Figure 2. Tear levels of tryptase were reduced significantly (P < 0.01) 5 minutes after allergen challenge in eyes treated with lodoxamide trometha­mine 0.1% when compared with eyes treated with placebo.

histamine-tryptase ratios were found in conjunctival ep­ithelial cell suspension of children with allergic conjuncti­vitis.14 We measured the protective effect of lodoxamide on mast cells by measuring tryptase in the tear fluid. Our study showed that lodoxamide significantly reduces this mast cell mediator in the tear fluid after allergen chal­lenge.

The presence of inflammatory cells in the tear fluid is consistent with an immediate hypersensitivity reaction after allergen challenge and has been associated with the occurrence of a late phase reaction. 15

'16 In fact, inflamma­

tory cells and mast cell-derived products may, in turn, activate other cells or toxic products responsible for chronic inflammation and hypersensitivity reaction. In particular, eosinophils are active cells in ocular allergic inflammation. 17 Their presence is justified by chemotactic activity of other mediators and is associated with the ocu­lar signs observed clinically in ocular allergies. We found that lodoxamide significantly decreased neutrophil and eosinophil accumulation in the tear fluid during the imme­diate hypersensitivity reaction.

In conclusion, our study supports previous findings on the efficacy of lodoxamide in the treatment of allergic conjunctivitis and shows that these clinical effects are associated with an objective inhibition of inflammatory cells and mediators in the tear fluid.

Bonini et al · Lodoxamide in Allergic Conjunctivitis

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8. Yanni JM, Weimer LK, Glaser RL, et al. Effect of lodoxa­mide on in vitro and in vivo conjunctival immediate hyper­sensitivity responses in rats. Int Arch Allergy Immunol 1993; 101:102-6.

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10. Fahy G, Easty DL, Collum L, et al. Double masked efficacy and safety evaluation oflodoxamide 0.1% ophthalmic solu­tion versus Opticrom 2% - A multicentre study. In: Ferraz LN, ed. Ophthalmology Today: Proceedings at the VIIIth Congress at the European Society at Ophthalmology, 1988 May 16-20. Lisbon, Portugal, Amsterdam, New York: Ex­cerpta Medica; New York, NY: distributors for the USA and Canada, Elsevier Science Pub. Co., 1988;341-2.

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